Members of the CIC family orchestrate the movements of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. Although certain eukaryotic CIC channels have been well characterized functionally, we remain in the dark about their detailed structures. Conversely, the structures of two prokaryotic CIC homologs were determined by x-ray crystallography, but we know little about their function. The high sequence identity between core invariant segments of prokaryotic and eukaryotic CICs argues for a shared structural scaffold, but the low (15-20%) overall identity also indicates that these families; differ in significant details. A striking functional correlate of the structural differences is the recent finding that CIC-ec1, an E. coli CIC, is not a chloride channel but a chloride-proton antiporter. Thus, the CIC family straddles an evolutionary cusp between these two different transport mechanisms. Our long-range goal is to determine how proton and chloride movement are coupled in CIC-ec1 and how this relates to proton- and chloride-activation of voltage-dependent gating in CIC-0. This work will shed light on the general mechanism of chloride permeation in the CIC family members, and will be essential for understanding the diverse physiology and pathophysiology seen amongst the CIC family members. In Specific Aim 1, we will test models of chloride-proton antiport using bilayer recordings and flux assays together with site-directed mutagenesis. Important technical developments include methods for obtaining oriented transporters, and the use of a high-throughput fluorescence-based transport assay to screen for specific inhibitors. In Specific aim 2, we will map the intracellular vestibule of CIC-0 using cysteine scanning mutagenesis. We will probe gating conformational changes by examining rates of cysteine modification under different conditions. Finally, we will analyze voltage- and proton-dependent gating at the single-channel level for mutations that cause significant changes in gating. Together, these studies will offer new insights into the functional relationships between channels and transporters.